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Nuclear Medicine and Biology 36 (2009) 155 – 162
www.elsevier.com/locate/nucmedbio

Preparation and evaluation of ethyl [18 F]fluoroacetate as a proradiotracerof [18 F]fluoroacetate for the measurement of glial metabolism by PET
Tetsuya Moria , Li-Quan Suna,b , Masato Kobayashia , Yasushi Kiyonoa,⁎, Hidehiko Okazawaa ,Takako Furukawaa , Hidekazu Kawashimac , Michael J. Welchd , Yasuhisa FujibayashiaaBiomedical Imaging Research Center, University of Fukui, Fukui 910-1193, Japan
School of Life Science and Technology, Beijing Institute of Technology, Beijing 100081, Chinac
Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japand
Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA
Received 3 October 2008; received in revised form 5 November 2008; accepted 11 November 2008b

Abstract
Introduction: Changes in glial metabolism in brain ischemia, Alzheimer's disease, depression, schizophrenia, epilepsy and manganeseneurotoxicity have been reported in recent studies. Therefore, it is very important to measure glial metabolism in vivo for the elucidation anddiagnosis of these diseases. Radiolabeled acetate is a good candidate for this purpose, but acetate has little uptake in the brain due to its low
lipophilicity. We have designed a new proradiotracer, ethyl [18F]fluoroacetate ([18F]EFA), which is [18F]fluoroacetate ([18F]FA) esterified
with ethanol, to increase the lipophilicity of fluoroacetate (FA), allowing the measurement of glial metabolism.
Methods: The synthesis of [18F]EFA was achieved using ethyl O-mesyl-glycolate as precursor. The blood–brain barrier permeability of ethyl[1-14C]fluoroacetate ([14C]EFA) was estimated by a brain uptake index (BUI) method. Hydrolysis of [14C]EFA in the brain was calculated by
the fraction of radioactivity in lipophilic and water fractions of homogenized brain. Using the plasma of five animal species, the stability of[14C]EFA was measured. Biodistribution studies of [18F]EFA in ddY mice were carried out and compared with [18F]FA. Positron emission
tomography (PET) scanning using common marmosets was performed for 90 min postadministration. At 60 min postinjection of [18F]EFA,
metabolite studies were performed. Organs were dissected from the marmosets, and extracted metabolites were analyzed with a thin-layer
chromatography method.
Results: The synthesis of [18F]EFA was accomplished in a short time (29 min) and with a reproducible radiochemical yield of 28.6±3.6%(decay corrected) and a high radiochemical purity of more than 95%. In the brain permeability study, the BUI of [14C]EFA was 3.8 times
higher than that of sodium [1-14C]fluoroacetate. [14C]EFA was hydrolyzed rapidly in rat brains. In stability studies using the plasma of fiveanimal species, [14C]EFA was stable only in primate plasma. Biodistribution studies in mice showed that the uptake of [18F]EFA in selectedorgans was higher than that of [18F]FA. From nonprimate PET studies, [18F]EFA was initially taken into the brain after injection. Metabolites
related to the tricarboxylic acid (TCA) cycle were detected in common marmoset brain.
Conclusion: [18F]EFA rapidly enters the brain and is then converted into TCA cycle metabolites in the brains of common marmosets. [18F]EFA shows promise as a proradiotracer for the measurement of glial metabolism.
© 2009 Elsevier Inc. All rights reserved.
Keywords: [18F]Fluoroacetate; Ethyl [18F]fluoroacetate; Proradiotracer; Glial metabolism; PET

1. Introduction
There is now a growing body of evidence indicating thatglial cells, particularly astrocytes, contribute actively tosynapse development, synaptic transmission and neuronal⁎ Corresponding author. Tel.: +81 776 61 8420; fax: +81 776 61 8170.
E-mail address: ykiyono@u-fukui.ac.jp (Y. Kiyono).
0969-8051/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.nucmedbio.2008.11.006

excitability [1–4]. Furthermore, changes in glial metabolism
in brain ischemia [5,6], Alzheimer's disease [7], depression[8], schizophrenia [9], epilepsy [10] and manganeseneurotoxicity [11] have been reported. Therefore, it is very
important to measure glial metabolism in vivo for the
elucidation and diagnosis of these diseases.
For this purpose, a positron emission tomography (PET)tracer for measuring glial metabolism should provide useful

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information. Radioactive carbon-labeled acetate has beenused for glial metabolism studies in basic research because exogenous acetate is selectively taken up by astrocytes via monocarboxylic acid transporter-1 and isconverted into acetyl-CoA in the cells [12,13]. However,the brain uptake of acetate from the blood stream is very
low due to low lipophilicity.
To resolve this problem, two approaches have recently
been reported. One approach using [1-11 C]octanoate has
been reported by Kuge et al. [14,15] and Yamazaki et al.
[16] Their strategy was to use high lipophilic fatty acid forblood–brain barrier (BBB) permeability and to measure
β-oxidation activity in astrocytes. Another approach was aproradiotracer concept reported by Inoue et al. [17] andMomosaki et al. [18] Their strategy involved combining
radiolabeled acetate with a highly lipophilic group for BBB
permeability, and then this proradiotracer is cleaved toradiolabeled acetate and the lipophilic group in the brain bycarboxyesterase. However, both approaches have limited
use because both compounds were labeled with carbon-11(half-life, 20 min).
We have designed a new proradiotracer, ethyl [18 F]
fluoroacetate ([18 F]EFA), which is esterified [18 F]fluoroacetate ([18 F]FA) with ethanol (Fig. 1). [3 H]Fluoroacetate
has been reported to accumulate selectively in glia and has
been proposed as potential tracer for glial oxidative
metabolism [12]. Fluoroacetate (FA) has been extensively
used in brain metabolism studies as a specific inhibitor of
glial cell metabolism [19,20]. Moreover, Hassel et al. [21]demonstrated that FA did not inhibit neuronal tricarboxylic
acid (TCA) cycles, but inhibited glial TCA cycles in miceusing nuclear magnetic resonance spectroscopy and gas
chromatography/mass spectroscopy. Therefore, FA isconsidered to be selectively transported into the glial
metabolic compartment and converted into fluoroacetyl-Fig. 1. The concept of a proradiotracer of [18F]FA for assessing glial
metabolism in the brain. FA: fluoroacetate; EFA: ethyl fluoroacetate.

CoA and 2-fluorocitrate in TCA cycles [12,20,22].
Fluorine-18 has a half-life (110 min) longer than that ofcarbon-11, is more convenient for the synthesis andmetabolism studies of PET radiopharmaceuticals, and canalso able be applied commercially [23].
In this study, the possibility of [18F]EFA as a proradiotracer
for the measurement of glial metabolism was assessed byinvestigating membrane permeability, ester hydrolysis in thebrain and plasma, and PET imaging in common marmosets.
2. Materials and methods
2.1. Reagents, instruments and materials
All chemicals were obtained from commercial sources andused without further purification. The following labeled
compounds, ethyl [1-14C]fluoroacetate ([14C]EFA), [1-14C]
fluoroacetate ([14C]FA) and [3H]H2O, were purchased from
American Radiolabeled Chemicals, Inc. (St. Louis, MO,
USA), and [1,5-14C]citrate and [1-14C]acetyl-CoA werepurchased from Amersham Biosciences, Inc. (UK). Ethyl
glycolate and methanesulfonyl chloride were obtained from
Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan). Ethyl
fluoroacetate, as reference standard, was purchased from
Wako Pure Chemical Industries, Ltd. (Tokyo, Japan).
Potassium carbonate, sodium hydroxide, anhydrous magnesium sulfate, hydrochloric acid, anhydrous acetonitrile andmethylene chloride were purchased from Sigma-Aldrich
Japan KK (Tokyo, Japan). Triethylamine and sodium
bicarbonate were purchased from Kanto Kagaku (Osaka,
Japan). Ethanol [99.5% for high-performance liquid chromatography (HPLC)] was purchased from Nacalai Tesque,Inc. (Kyoto, Japan). Sep-Pak cartridges were purchased from
Waters Corporation (Milford, MA, USA).
Analytical HPLC was performed on a Waters Corporation
600E system equipped with Waters 490E programmablemultiwavelength UV detector set at 210 nm and a Bioscan
Flow counter (Washington, DC, USA). A reversed-phase
Hydrosphere C18 column (4.6×150 mm; YMC, Kyoto,
Japan) was used with ethanol/H2O (10:90) as mobile phaseat a flow rate of 1.0 ml/min. Radio thin-layer chromatography (TLC) was performed using 0.25-mm Silica Gel 60
Sheets F254 purchased from Nacalai Tesque, Inc.
Plasma from rats and rabbits was obtained from Sankyo
Labo Service Co. (Tokyo, Japan), and dog plasma was
acquired from NARC Co. (Chiba, Japan). Common
marmosets (Callithrix jacchus) weighting 340–390 g wereobtained from Japan SLC, Inc. (Shizuoka, Japan), andplasma was prepared from fresh whole blood. Humanplasma was prepared from fresh whole blood provided by ahealthy volunteer.
Wistar rats and ddY mice were purchased from Sankyo
Labo Service Co. and maintained in a specific pathogen-freeroom. Common marmosets were housed in stainless-steel
cages in a temperature-controlled (28±2°C) and humiditycontrolled (50±10%) animal room and fed a balanced

T. Mori et al. / Nuclear Medicine and Biology 36 (2009) 155–162

marmoset diet (CMS-1; CLEA Japan, Inc., Tokyo, Japan)
with free access to water. Animal experiments were carriedout in accordance with the Institutional Guidelines for theCare and Use of Experimental Animals and approved by theinternal ethical committees of the Department of Medicine,
University of Fukui.
2.2. Preparation of no-carrier-added [18F]EFA and [18F]FA
Synthesis of [18F]EFA was performed using a simple
instrument set-up, as shown in Fig. 2A. In this reaction,
ethyl O-mesyl-glycolate was synthesized first, then it wasused as precursor (Fig. 2B) [23]. No-carrier-added [18F]
fluoride was produced via the 18O(p,n) 18F reaction in a
Siemens RDS eclipse cyclotron (11 MeV protons) on anenriched water target. No-carrier-added aqueous [18 F]
fluoride (1.35 ml; 3.7 GBq) was added onto a Sep-Pak
Acell QMA Light cartridge, which was converted into CO2−
3
form by treatment of an aqueous solution of 0.1 M K2CO3.
Almost all [18F]fluoride anions were extracted from [18O]
water and trapped by the cartridge, and the enriched [18O]
water was recovered in the reservoir. Half a milliliter of 16.7
mM aqueous potassium carbonate solution was employed
to elute the trapped [18F]fluoride anion from the QMA

157
18

cartridge. The eluate with [ F]fluoride solution was
introduced to the reaction vessel. The solution of 16.4 mg
of Kryptofix 2.2.2 dissolved in 1 ml of anhydrous
acetonitrile was then added to the reaction vessel. Water inthe mixture was azeotropically evaporated three times at
95°C with nitrogen gas flow and anhydrous acetonitrile(500 μl×3). After the drying step, 2.9 mg of ethyl O-mesylglycolate dissolved in 1 ml of anhydrous acetonitrile wasadded to the residue in the reaction vessel. The labelingreaction was maintained at 105°C for 5 min in the closedreaction vessel to give rise to [18F]EFA. Then, the reaction
solvent anhydrous acetonitrile was removed at 95°C
with nitrogen gas flow. After 5 min, the residue in thereaction vessel was distilled under reduced pressure (about
14 mmHg), and the distillate [18F]EFA was collected andtrapped by 1 ml of ice-cold ethanol (99.5%) in a 3-ml vial.
Finally, the ethanol solution was transferred through a SepPak Alumina N Plus cartridge and further filtered by a0.22-μm sterile filter. The product was analyzed by TLC and
HPLC. When animal experiments were performed, the final
product was diluted with saline to 5% ethanol solution. The
preparation of [18F]FA was performed as described previously by Sun et al. [23].

Fig. 2. (A) Diagram of a simple instrument for the preparation of [18F]EFA. (B) Scheme of [18F]EFA synthesis.

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14

2.3. Brain permeability studies of [ C]EFA
14

14

The brain uptakes of [ C]EFA and sodium [ C]FA
were estimated by the brain uptake index (BUI) method[24]. Male Wistar rats weighing 230–250 g (n=4) wereanaesthetized using sodium pentobarbital (35 mg/kg ip),
and their right carotid arteries were surgically exposed.
Saline solution (0.1 ml) containing 37 kBq of carbon14-labeled compound and 37 kBq of [3H]water was
injected via the carotid artery. At 20 s after injection, therat was decapitated, and its brain was removed. The rightcortex was weighed (50 mg), then 1 ml of Soluene (PerkinElmer, Inc., Waltham, MA, USA) was added for liquefaction. After 1 h of incubation at 60°C, 9 ml of cocktail wasadded to the sample, and radioactivity was counted in aliquid scintillation counter (Packard Instruments Co., VA,
USA). The BUI was calculated according to Eq. (1):h14BUIð%Þ ¼

i
C in the right cortex=½3 H in the right cortex  100h
14C injected =½3 H injected
ð1Þ

2.4. Hydrolysis studies in rat brain
Male Wistar rats (n=3) were anaesthetized with an
intraperitoneal injection of sodium pentobarbital, and acarotid artery was surgically exposed. [14C]EFA (37 kBq) in
0.1 ml of saline was injected via the carotid artery, and the rat
was decapitated at 5 min after the injection. The cerebral
cortex (50 mg) was homogenized in 0.5 ml of ice-cold
10 mM Tris–HCl buffer (pH 7.4) containing 0.25 M sucroseand extracted with 0.5 ml of ethyl acetate. The radioactivitiesof the lipophilic and water fractions were counted by a liquid
scintillation counter. The percentage of radioactivity in the
lipophilic fraction was calculated by dividing the radioactivity in the lipophilic fraction by the sum of both fractions,as shown in Eq. (2):k of 14C in lipophilic fraction
14C in lipophilic fraction
 100=
14C in lipophilic fraction + 14C in water fraction

were collected (50 μl), respectively. The radioactivity in eachfraction was counted by a liquid scintillation counter. All
incubations were performed in triplicate. The percentage
of radioactivity in the lipophilic fraction was calculated in
Eq. (2).
2.6. Biodistribution studies in ddY mice
Biodistribution studies of [18F]EFA and [18F]FA werecarried out using 7-week-old ddY mice. Each compound(37 kBq) was intravenously injected and sacrificed at 30, 60
and 90 min postinjection (n=3-4 per time point). Samples ofblood, brain, heart, liver, bone and muscle were removed andweighed. Their radioactivity was counted in an Aloka ARC380 well scintillation counter (Aloka, Tokyo, Japan). All data
were corrected for 18F decay and reported as percent injected
dose per gram of tissue (%ID/g ± S.D.)
2.7. PET imaging studies in common marmosets
PET imaging studies were performed with an ADVANCE
system (General Electric Medical System, Milwaukee, WI).
The physical characteristics of this scanner have been
described in detail by DeGrado et al. [25]. A standard pin
source of 68Ge/68Ga was used for attenuation corrections ofemission images. Common marmoset was anesthetized witha ketamine–xylazine cocktail (20 and 3 mg/kg im,
respectively) and sodium pentobarbital (20 mg/kg ip) [26].
[18F]EFA (11.9–12.6 MBq; n=3) or [18F]FA (13.8 MBq;n=1) was injected via a saphenous vein. PET scanning was
performed for 90 min with 12 frames at 10-s intervals, 8frames at 1 min, 4 frames at 5 min, and 6 frames at 10 min.
After the scanning, a 6-mm circular region of interest (ROI)
was placed on transaxial PET images at the area of thehighest accumulated radioactivity of [18F]EFA in the brain.
The ROI for [18F]FA was placed on the same area of [18F]
EFA. The percent injected dose per gram of ROI (%ID/g) for
time–activity curves was calculated using Eq. (3). In Eq. (3),a calibration factor was applied to these ROI data:

ð2Þ

tissue radioactivity concentration ðMBq=mlÞ
injection dose ðMBqÞ
tissue volume ðmlÞ
 100
ð3Þ

body weight ðgÞ

2.5. Stability studies in the plasma of several animal species

2.8. Metabolite studies of [18F]EFA in nonhuman primate

The hydrolysis of radiolabeled EFA in the plasma wasinvestigated using five animal species, namely, rat, rabbit,
dog, common marmoset and human (n=3, respectively).
Each plasma sample (200 μl) was incubated with [14C]EFA(370 Bq/10 μl in ethanol) at 37°C in a 1.5-ml microcentrifuge
tube. After 5, 10, 30 and 60 min, the hydrolysis reactions
were stopped by adding ice-cold water-saturated ethyl
acetate (200 μl). The mixture was vortexed and centrifuged
at 14,000 rpm for 1 min. The ethyl acetate fraction of theintact form and the water fraction of hydrolyzed metabolites

Common marmosets (n=2) were anesthetized, and [18F]EFA was injected via a saphenous vein. At 1 h afteradministration, the marmoset was euthanized, and arterial
blood and selected organs (namely, brain, heart, lungs, liverand kidneys) were collected. The organs were homogenized
by a polytron homogenizer with 10 times volume of ice-coldmethanol and then centrifuged at 15,000 rpm for 10 min. The
supernatants thus obtained were spotted on a TLC plate anddeveloped with standards, namely, [18F]EFA, [18F]FA, [14C]
citrate, [14C]acetyl-CoA and [18F]NaF, using the following%ID=g ¼

T. Mori et al. / Nuclear Medicine and Biology 36 (2009) 155–162

159
14

3.2. Brain permeability studies of [ C]EFA
Brain permeability was estimated by the BUI method
using 14C compounds and [3H]H2O. The BUIs of [14C]EFA
and [14C]FA were 125.9±4.1% and 33.3±3.7%, respectively(n=4). These data show that the BUI of [14C]EFA was 3.8
times higher than that of [14C]FA.
3.3. Hydrolysis studies in rat brain
In vivo hydrolysis of [14C]EFA was estimated using rat
brain. The percentage of radioactivity in lipophilic and water
fractions was calculated after extraction from the cerebral
cortex. The percentage of radioactivity in the lipophilic
fraction representing the intact [14C]EFA was 3.5±3.0% of
the total radioactivity at 5 min after injection (n=3).
3.4. Stability studies in the plasma of several animal species

Fig. 3. The percentage of intact [14C]EFA against time in the plasma of fiveanimal species.

solutions: (a) chloroform/methanol (1:1) and (b) diethyl
ether/formic acid/water (3:1:1). The former condition could
separate [18F]EFA (Rf=0.9), [18F]FA (Rf=0.7) and others(Rf=0). The latter one distinguished among [14C]citrate(Rf=0.3), [14C]acetyl-CoA (Rf=0) and others (Rf=0.9). [14C]
citrate and [14C]acetyl-CoA were used as standards todetermine [18 F]fluorocitrate and [18 F]fluoroacetyl-CoA,
respectively. Metabolites were detected using a Fuji Photo
Film BAS-1500 imaging analyzer (Fuji, Tokyo, Japan) andidentified by comparison with standards.
3. Results
3.1. Synthesis of [18F]EFA[18F]EFA was obtained from ethanol solution with a yieldof 28.6±3.6% (n=11). Radiochemical purity was determined
to be up to 95%, and synthesis time was 29 min. The stabilityof [18F]EFA was confirmed after 6 h.

The stability of radiolabeled EFA in plasma wasinvestigated using several animal species. The time coursesof the intact [14C]EFA in plasma are given in Fig. 3. In both
rat and rabbit plasma, [14C]EFA was hydrolyzed quickly, andless than 21% of the radioactivity was found in the lipophilic
fraction at 30 min. Dog plasma showed a gradual degradation curve, and about 58% of the radioactivity was extracted
in lipophilic fraction at 60 min. In primates (namely,
marmosets and humans), over 80% of the radioactivity was
found in the lipophilic fraction at 60 min.
3.5. Biodistribution studies in ddY mice
The biodistribution of [18 F]EFA and [18 F]FA wascarried out in ddY mice. Table 1 displays the percent
injected dose per gram of tissue (%ID/g ± S.D.) of selectedorgans at 30, 60 and 90 min after injection. Intense
activities were found in the blood, heart and lungs in [18 F]
EFA and [18 F]FA. We also confirmed brain uptake in bothtracers, and the %ID/g value of [18 F]EFA was higher thanthat of [18 F]FA at 30 min (6.88±0.53%ID/g vs. 4.97±0.38%
ID/g, respectively; P=0.001). In the case of [18 F]FA, slow
clearance from the brain was observed from 30 to 90 min(4.97±0.38%ID/g and 4.05±0.22%ID/g, respectively;
P=0.006); however, the activity in the brain did not change

Table 1
Biodistribution data of [18F]EFA and [18F]FA in ddY mice (n=3 or 4)
Organ

Blood
Brain
Heart
Lungs
Liver
Bone
Brain/blood ratio[18F]EFA (%ID/g±S.D.)[18F]FA (%ID/g±S.D.)

30 min60 min90 min30 min60 min90 min10.88±0.62⁎⁎
6.88±0.53⁎⁎
10.66±1.67⁎⁎
9.94±1.14⁎⁎8.42±0.36⁎⁎
8.55±1.16⁎
0.63±0.03

9.46±0.80⁎⁎
6.41±0.39⁎
10.92±1.17⁎⁎
8.63±0.53⁎⁎
7.48±0.29⁎⁎
10.66±2.71
0.68±0.05

8.51±0.49⁎⁎
6.37±0.11⁎⁎
8.93±1.64⁎
7.94±0.35⁎⁎
9.04±3.44⁎
11.42±3.85
0.75±0.06

7.10±0.72
4.97±0.38
6.12±1.08
5.70±0.88
5.37±0.54
4.64±1.78
0.70±0.04

7.46±0.33
5.13±0.55
7.56±0.84
6.17±0.31
5.49±0.54
10.27±2.60
0.69±0.06

6.08±0.47
4.05±0.22
5.93±0.80
5.22±0.49
4.61±0.64
8.22±2.25
0.67±0.03⁎ Pb0.05 compared with [18F]FA data in the corresponding tissue.
⁎⁎ Pb0.01 compared with [18F]FA data in the corresponding tissue.

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18

from 30 to 90 min in [ F]EFA (6.88±0.53%ID/g and6.37±0.11%ID/g, respectively; P=ns). The radioactivity inthe bone, indicating defluorination, increased significantly
over time following the administration of both compounds.
3.6. PET studies of [18F]EFA and [18F]FA
A brain PET image of [18F]EFA and [18F]FA in acommon marmoset is shown in Fig. 4A. The time–activity
curves of ROIs for [18F]EFA and [18F]FA in the brain are
shown in Fig. 4B. The radioactivity of [18F]EFA in the brainincreased to 0.29±0.07%ID/g at 3 min after injection andthen decreased gradually up to 10 min. After that, theradioactivity was retained until 90 min, and the %ID/g value
was higher than that of [18F]FA (0.22±0.03%ID/g vs. 0.18%
ID/g at 60 min, and 0.21±0.03%ID/g vs. 0.17%ID/g at
90 min, respectively).

Fig. 5. Radio-TLC analysis of the metabolites in common marmoset at 1 h
after injection of [18F]EFA (Solvent B). The Rf values at this condition are as
follows: [18F]EFA: 0.9; [18F]FA: 0.9; [14C]citrate: 0.3; [14C]acetyl-CoA: 0.0;
[18F]NaF: 0.9.

3.7. Metabolites in marmosets
Fig. 5 shows the radio-TLC of metabolites in typicalorgans. The metabolites in the whole blood, liver andkidneys at 1 h after administration were only [18F]FA, but
unidentified metabolites were detected near the [14C]citrate's
Rf value in the brain, heart and lungs.
4. Discussion

Fig. 4. (A) Dynamic brain PET images of [18F]FA (left) and [18F]EFA (right)(0–10 min) in nonhuman primate. The images are fused to T1-weighted
magnetic resonance images. ROI in the brain set in the black circle. (B)Time–activity curves of [18F]EFA-PET (n=3) and [18F]FA-PET (n=1) in
common marmoset. The values for [18F]EFA are given as mean±S.D. fromthree animals. Common marmoset was anesthetized with a ketamine–
xylazine cocktail (20 and 3 mg/kg im, respectively) and sodium
pentobarbital (20 mg/kg ip). [18F]EFA (11.9–12.6 MBq; n=3) or [18F]FA(13.8 MBq; n=1) was injected via a saphenous vein. PET scanning was
performed for 90 min with 12 frames at 10-s intervals, 8 frames at 1 min, 4frames at 5 min, and 6 frames at 10 min.

We selected [18F]EFA, instead of radiolabeled acetate, as
an imaging tracer for glial metabolism in view of themembrane permeability and characteristics of ester compounds. This ester is considered to offer high membrane
permeability, and ethanol is safe to tissues after thehydrolysis of acetate ester.
The synthesis of [18F]EFA was performed by a facile
method via distillation on a simple instrument set-up. The
instrument did not need special parts and could be assembled
in any chemical laboratory. This synthesis might be performed
anywhere even without an automatic synthesizer. As [18F]EFA is not stable in aqueous solution, we attempted to trap itwith pure ethanol in distillation. At the same time, ethanol wascooled by ice water. Before use in animal experiments, theethanol solution might immediately be diluted by saline. This
procedure offers a good way of providing a high concentrationof [18F]EFA at the time of injection.
The BUI studies revealed that radiolabeled EFA has highBBB permeability, the value being 3.8 times higher than thatof FA. This result suggested that our strategy was appropriate
in view of the BBB permeability. To evaluate ourproradiotracer concept, the percentage of intact [14C]EFA
was calculated. The result showed that the intact [14C]EFA
was 3.5±3.0% in the rat cortex at 5 min after injection. It issuggested that [14C]EFA was hydrolyzed rapidly in the brain

T. Mori et al. / Nuclear Medicine and Biology 36 (2009) 155–162

and that EFA functioned as a proradiotracer based on our
strategy. However, it is known that esterase activity in the
blood varies considerably among animal species [27–30], so
the stability of EFA was assessed in the plasma of five animal
species. These experiments demonstrated that [14C]EFA washydrolyzed instantly in rodent plasma; however, it was stablein primate plasma (Fig. 3).
The biodistribution studies in mice were carried out tocompare tissue uptake and clearance between [18F]EFA and[18F]FA. The uptake of [18F]EFA in the brain demonstrated
retention in the brain and faster clearance in the blood at 30
and 90 min after injection, compared to [18F]FA. This may
be due to the difference in lipophilicity of the two tracers,and [18F]EFA would be available for delayed brain imaging.
The uptakes of [18F]EFA in the heart and lungs were alsohigh. These high uptakes suggest that [18F]EFA may also be
useful for myocardial and pulmonary perfusion imaging.
Intense accumulation in the bone, which indicates defluorination, was observed over time in both tracers. We believethat the defluorination was derived from [18F]FA because
Ponde et al. [31] reported that [18 F]FA was rapidly
decomposed in rodents. In these biodistribution studies, wefound differences in in vivo behavior between the two
tracers. However, we were unable to disregard two points asfollows: the difference in the ester stability and defluorination of [18F]FA between animal species. In the former point,
we confirmed that [14C]EFA was more stable in primate
plasma than in rodent plasma. In the latter, Ponde et al. [31]also reported that [18F]FA was stable in primates for 2 h.
Therefore, we performed PET and metabolism studies using
nonhuman primate (namely, common marmoset) in order toassess [18F]EFA as a proradiotracer for the measurement ofglial metabolism.
In nonhuman primate PET studies, a high accumulation ofradioactivity in the brain was observed when [18F]EFA wasinjected (Fig. 4A). The radioactivity in the brain increased to
0.29±0.07%ID/g at 3 min after injection and then decreased
quickly to 10 min (Fig. 4B). After 10 min, sufficientradioactivity was retained in the brain. However, theequilibrium in the brain was significantly higher following[18F]EFA administration than following [18F]FA administration, but was not as great as the difference at early time
points in the primate. The first uptake until 10 min may
represent BBB permeability depending on blood flow, andclearance from the brain may represent competition between
the rate of ester hydrolysis and the reverse BBB permeabilityof [18F]EFA. Furthermore, since the metabolic rate of FA isslow [20], the hydrolyzed [18F]EFA (i.e., [18F]FA) also could
gradually clear from the brain. The gradual decrease inradioactivity until about 30 min may represent this condition.
After 30 min, the retention may represent the trapped [18F]EFA such as [18F]FA and/or its metabolite in the TCA cycle.
These results shows that [18F]EFA could function as aproradiotracer of [18F]FA in nonhuman primate.
We confirmed that the uptake of [18 F]EFA in the brain ishigher than that of [18 F]FA in both primates and rodents.

161

They were normal animals, and so the amount of retention
will be different in disease states where glial metabolism
is altered. It is highly likely that, due to the greater deliveryof [18 F]EFA to the brain compared to [18 F]FA, thedifference in these disease areas will be greater than thatin the normal brain.
The metabolism studies in common marmosets revealed
that only [18F]FA was detected in the blood, liver and kidney
at 1 h after injection, as shown in Fig. 5. Interestingly,
metabolites such as [18F]FA and [18F]fluorocitrate were
determined in the heart, lungs and brain. These results
suggested that the metabolism of [18F]EFA via the TCA
cycle in the brain occurred through the oxidative route andwas more rapid than that in other organs. It was reported thatthe metabolites of [14C]FA in rat brains were practically
undetectable at 10 min and 1 h [20]. However, metabolites
were confirmed at 1 h in primate brains in our experiments. It
is known that the symptoms of FA toxicity are different
among animal species [32], so the metabolic pattern(metabolic rate, amount, formation and so on) of EFAmight be dependent on species. Further studies of metabolites in the brain are required.
5. Conclusion[18 F]EFA rapidly entered the brain and then washydrolyzed to TCA cycle metabolites in the brains ofcommon marmosets. [18F]EFA is a candidate proradiotracer
for the measurement of glial metabolism in the brain,
although further studies are required to analyze themetabolites in the brain.
Acknowledgments
Part of this study was supported by the Research andDevelopment Project Aimed at Economic Revitalizationfrom the Ministry of Education, Culture, Sports, Science andTechnology Japan, the 21st Century COE program “Biomedical Imaging Technology Integration Program” from theJapan Society for the Promotion of Science, and a grant for
Collaboration of Regional Entities for the Advancement ofTechnological Excellence from the Japan Science andTechnology Agency.
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